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Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Sandra Vergara,Xiaohong Zhou,Ulises Santiago,Mounia Alaoui-El-Azher,James F. Conway,Nicolas Sluis-Cremer,Guillermo Calero

Published 2024 in Nature Communications

ABSTRACT

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication. The intermediate states occurring during nucleotide addition by HIV-1 RT remain unclear. Here, authors use cryo-EM to capture five unique states that show how a mobile catalytic Mg2+ drives phosphodiester bond formation.

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